Establishing coordinate gene regulation during Drosophila dosage compensation

描述（由申请人提供）：所有生物体在正常发育期间必须协调调节其基因，以预防疾病状态。然而，基因被确定为协调调节的机制仍然知之甚少。我们的长期目标是描述对靶基因进行协调调控至关重要的大分子相互作用，这是其调控的关键初始步骤。剂量补偿是研究这一过程的最佳模型系统之一，因为单个染色体上的所有基因都被特异性识别和共同调控。果蝇与哺乳动物一样，沿着单个雄性X染色体的长度，相对于每个雌性X染色体，增加了沿着大量双调节基因的转录水平[2]。该应用程序的目的是生成第一个三维模型的剂量补偿在果蝇是如何建立的，在监管过程中的关键的第一步。果蝇雄性特异性致死（MSL）复合物是剂量补偿的核心;它首先使用顺式作用DNA序列和其roX（X上的RNA）非编码RNA组分的共转录募集的组合来识别X染色体，然后扩散到活性基因的体内。然而，我们不知道MSL复合物如何特异性地识别雄性X上的MSL识别元件（MRE）序列，因为已知的MSL组分不足以在体外直接识别MRE。我们进行了一项创新的基因筛选，寻找在男性和女性中发挥作用的剂量补偿新调节剂，从而确定了以前未研究的CLAMP锌指蛋白。在强有力的初步数据的指导下，我们提出了以下用于鉴定用于协调调节的基因的新机制：CLAMP蛋白质集中于细胞核内的病灶最初将MSL复合物靶向X染色体，随后两个因子之间的协同相互作用增加了它们的占据。这项工作的基本原理是，我们的CLAMP蛋白的鉴定提供了一个重要的机会，以了解所需的大分子相互作用，以确定一个亚核结构域的增强转录。我们将使用两个新的具体目标来测试这种新的机制：目标#1：通过使用染色体构象捕获来定义X染色体上的种子位点如何在细胞核内的三维结构;目标#2：使用多光子显微镜在三维中确定早期胚胎发生期间细胞核内CLAMP定位的动态。我们提出的研究是重要的，因为我们希望产生第一个三维模型，如何在整个染色体上的转录活性域形成。我们的三维模型将使我们能够区分两种长期存在的亚核结构域形成机制：1）不连续扩散，涉及初始种子位点的三维聚类; 2）连续扩散，涉及沿X染色体长度沿着扫描。确定CLAMP如何产生增强转录的结构域，可能会提供关键的洞察如何识别跨物种的协调调控基因。

DESCRIPTION (provided by applicant): All organisms must coordinately regulate their genes during normal development and to prevent disease states. However, the mechanisms by which genes are identified for coordinate regulation remain poorly understood. Our long-term goal is to describe the macromolecular interactions that are critical to target genes for coordinate regulation, the key initial step in their regulation. Dosage compensation is one of the best model systems for studying this process because all of the genes on a single chromosome are specifically identified and co-regulated. Drosophila, like mammals, increase the transcript levels of a large number of diversely regulated genes along the length of the single male X-chromosome precisely two-fold relative to each female X-chromosome [2]. The objective of this application is to generate the first three-dimensional model of how dosage compensation in Drosophila is established, the critical first step in the regulatory process. The Drosophila Male Specific Lethal (MSL) complex is central to dosage compensation; it first identifies the X chromosome using a combination of cis-acting DNA sequences and cotranscriptional recruitment by its roX (RNA on X) non-coding RNA components, and then spreads into the bodies of active genes. However, we do not know how the MSL complex specifically identifies the MSL Recognition Element (MRE) sequences on the male X because known MSL components are insufficient for direct recognition of MREs in vitro. We performed an innovative genetic screen for new regulators of dosage compensation that function in both males and females and thereby identified the previously-unstudied CLAMP zinc-finger protein. Guided by strong preliminary data, we propose the following novel mechanism for identifying genes for coordinate regulation: Concentration of the CLAMP protein to foci within the nucleus initially targets MSL complex to the X- chromosome, followed by synergistic interactions between the two factors that increases their occupancy. The rationale for this work is that our identification f the CLAMP protein provides a critical opportunity to understand the macromolecular interactions required to define a sub-nuclear domain of enhanced transcription. We will test this novel mechanism using two new specific aims: Aim #1: Define how seed sites on the X-chromosome are structured in three dimensions within the nucleus by using Chromosome Conformation Capture; Aim #2: Determine the dynamics of CLAMP localization within the nucleus during early embryogenesis in three dimensions using multiphoton microscopy. Our proposed research is significant because we expect to generate the first three-dimensional model of how an active domain of transcription on an entire chromosome is formed. Our three-dimensional model will allow us to distinguish between two longstanding mechanisms for how a sub-nuclear domain is formed: 1) Discontinuous spreading that involves three-dimensional clustering of initial seed sites; 2) Continuous spreading that involves scanning along the length of the X-chromosome. Defining how CLAMP generates a domain of enhanced transcription is likely to provide key insight into how genes are identified for coordinate regulation across species.